Broad surveys of DNA viral diversity obtained through viral metagenomics of mosquitoes

Abstract

Viruses are the most abundant and diverse genetic entities on Earth; however, broad surveys of viral diversity are hindered by the lack of a universal assay for viruses and the inability to sample a sufficient number of individual hosts. This study utilized vector-enabled metagenomics (VEM) to provide a snapshot of the diversity of DNA viruses present in three mosquito samples from San Diego, California. The majority of the sequences were novel, suggesting that the viral community in mosquitoes, as well as the animal and plant hosts they feed on, is highly diverse and largely uncharacterized. Each mosquito sample contained a distinct viral community. The mosquito viromes contained sequences related to a broad range of animal, plant, insect and bacterial viruses. Animal viruses identified included anelloviruses, circoviruses, herpesviruses, poxviruses, and papillomaviruses, which mosquitoes may have obtained from vertebrate hosts during blood feeding. Notably, sequences related to human papillomaviruses were identified in one of the mosquito samples. Sequences similar to plant viruses were identified in all mosquito viromes, which were potentially acquired through feeding on plant nectar. Numerous bacteriophages and insect viruses were also detected, including a novel densovirus likely infecting Culex erythrothorax. Through sampling insect vectors, VEM enables broad survey of viral diversity and has significantly increased our knowledge of the DNA viruses present in mosquitoes.

Figure 1. Taxonomic classification of the metagenomic sequences from the three mosquito viromes.

A) Classification based on tBLASTx (E-value <0.001) against the Genbank non-redundant database. B) Breakdown of the viral sequences into four major categories: animal, plant, insect viruses (densoviruses and other insect viruses) and bacteriophages. Samples were obtained from 3 sites in San Diego: Buena Vista Lagoon (SD-BVL), River Bank (SD-RB), and Wild Animal Park (SD-WAP).

Figure 2. Classification of vertebrate and plant virus sequences present in the three San Diego mosquito viromes.

The family, host, and name of the most significant tBLASTx similarities in the Genbank non-redundant database are shown, with the colors representing the level of amino acid identity.

Figure 3. Genome organization and coverage of several putative virus genomes discovered in mosquito viromes.

A) Human papillomavirus 23 (HPV23), B) Novel anelloviruses, C) Novel viruses with unique genome organization. Open reading frames are highlighted on the genome map and the amount of coverage from the metagenomic reads of the sample the virus was identified in is shown in the center.

Figure 4. Neighbor joining tree based on the amino acid alignment of Mosquito VEM Papillomaviruses with the partial capsid protein L2 of representative HPV types.

Mosquito VEM Papillomavirus – SDRB AF shared similarity to a different region of capsid protein L2, but produced identical tree topography (data not shown). Papillomavirus sequences identified in mosquito virome SD-RB are indicated by arrows, all of which belong to the cutaneous groups.

Figure 5. Neighbor joining phylogenetic tree of anelloviruses constructed using the entire nucleotide sequence of ORF1.

Genbank accession numbers are shown in parentheses, and the hosts are indicated for any non-human sequences. The newly discovered anelloviruses from the mosquito viromes are indicated by arrows.

Figure 6. Neighbor joining phylogenetic tree of VEMCeDNV and other densoviruses based on alignment of the 720-bp partial NS1 gene nucleotide sequences.

The VEMCeDNV from the C. erythrothorax mosquitoes in sample SD-WAP is indicated with an arrow.